CN114174509A - Primer, double-stranded DNA production apparatus using the same, and double-stranded DNA production method - Google Patents
Primer, double-stranded DNA production apparatus using the same, and double-stranded DNA production method Download PDFInfo
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- CN114174509A CN114174509A CN202080054765.7A CN202080054765A CN114174509A CN 114174509 A CN114174509 A CN 114174509A CN 202080054765 A CN202080054765 A CN 202080054765A CN 114174509 A CN114174509 A CN 114174509A
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- primer
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- stranded dna
- dna
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6846—Common amplification features
Abstract
The present invention provides a primer for amplifying a nucleic acid, which has a structure represented by the following formula (1). (Here, A)1represents-S-, -S-or-Se-, B represents a base group, R1Represents a decomposable protecting group. It means a bond to a sugar of an adjacent nucleotide. ). The present invention also provides an apparatus for producing a double-stranded DNA, comprising: a forward primer and a reverse primer having a structure represented by formula (1); a PCR device for performing PCR of a plurality of cycles using the template DNA as a template to generate a double-stranded DNA having a 3' -end recess; a klenow fragment with a 3' end rendered a smooth end; and a light irradiation device for irradiating R1Deprotection to form an adherent terminus protruding from the 3' terminus.
Description
Technical Field
The present invention relates to a primer, an apparatus for producing a double-stranded DNA using the primer, and a method for producing a double-stranded DNA.
Background
In the field of molecular biology and the like, vectors obtained by integrating a target DNA into a host are used for gene recombination, transformation, and the like. In general, when the amount of the target DNA is small, it is amplified by Polymerase Chain Reaction (PCR) and then used. In PCR, a template DNA containing a target DNA sequence is used, and thermal denaturation and annealing are repeatedly performed for a plurality of cycles using a primer complementarily binding thereto, thereby amplifying the template DNA.
The amplification product of the template DNA amplified by PCR is directly blunt-ended, and needs to be treated in order to bind (link) to host DNA such as plasmid DNA. In general, as such a treatment, a restriction enzyme that cleaves a specific sequence is used, but in this method, since DNA that can be bound depends on the sequence of the cleavage site of the restriction enzyme, there is a problem of lack of versatility.
As a method not using a restriction enzyme, there are the following techniques: the 3 '-end and the 5' -end of the amplification product are used as adhesion ends (also referred to as an adhesion end, a overhang end, etc.), and the adhesion ends are similarly formed on the host side, and the two are ligated to construct a vector. For example, In recent years, a Gibson assembly (Gibson assembly) method, an In-Fusion method, a SLiCE method, and the like have been known. In these methods, the double-stranded DNA fragments each have the same sequence of about 15bp at the end, and one strand of the double strand is digested with exonuclease activity to generate an adhesive end, followed by ligation. In addition, In the Gibson assembly method, ligation was performed In vitro using Taq DNA ligase, while In-Fusion method ligation was performed using a repair system In E.coli.
In these methods, since an exonuclease is used as an enzyme, there are cases where the cost is high and the site specificity is deteriorated due to reaction conditions and the like, and it is difficult to form a quantitative adhesive end, so that the efficiency of the ligation reaction is low. Therefore, a seamless cloning method without using an enzyme was sought.
Therefore, a method of chemically preparing a DNA having an adhesive end has been developed, and as a primer for PCR used in this method, a primer described in patent document 1 is known. In the primer of this document, the base corresponding to the 3' -end of the base sequence of the non-complementary DNA portion is modified with a protecting group. The protecting group has a function of stopping DNA replication by DNA polymerase, and can be detached from the modified base by light irradiation treatment, alkali treatment, or the like. In this document, a base protecting group is introduced into a base of a primer using a substituent introducing agent for introducing a protecting group (substituent) into a biomolecule.
Documents of the prior art
Patent document
Patent document 1: international publication No. 2009/113709 (claim 1, claim 2, etc.).
Disclosure of Invention
Technical problem to be solved by the invention
In patent document 1, although the progress of DNA replication is stopped by the protective group moiety, the site specificity is low because the single-stranded DNA is not cleaved at a specific site to generate an adhesive end. Further, there are 4 types of bases such as adenine, guanine, cytosine and thymine in DNA, but in patent document 1, in order to introduce a protecting group into a base, it is necessary to introduce a protecting group by a method corresponding to the type of base, which is troublesome and costly in the production of a primer.
The purpose of the present invention is to provide a primer that can specifically cleave a single strand at a specific site and that can be produced at low cost. Another object of the present invention is to provide: an apparatus for producing a double-stranded DNA having an adhesive end using such a primer, and a method for producing a double-stranded DNA.
Means for solving the problems
The present inventors have conducted intensive studies to solve the above problems. As a result, a primer for introducing a decomposable protecting group into the sugar portion of a nucleoside via a specific linker was developed. Moreover, it has also been found that: the protective group is cleaved to cleave a single strand at the nucleoside moiety, thereby producing a double-stranded DNA having an attached end, and the present invention has been completed.
That is, the present invention is a primer characterized in that: the primer for amplifying a nucleic acid has a structure represented by the following formula (1).
(Here, A)1represents-S-, -S-or-Se-, B represents a base group, R1Represents a decomposable protecting group. It means a bond to a sugar of an adjacent nucleotide. )
In this case, R is preferably R1Is a photolytic protecting group represented by the following formula (2A).
(Here, A)2Represents an alkylene group having 1 to 3 carbon atoms, A3Represents an alkylene group having 1 to 3 carbon atoms. Means with A1The bond of (2). )
Further preferably R is the above-mentioned1Is a 2-nitrobenzyloxymethyl group represented by the following formula (3A).
Alternatively, the above-mentioned R is preferable1Is a fluoride-decomposable protecting group represented by the following formula (2B).
(Here, A)4Represents an alkylene group having 1 to 3 carbon atoms, R2～R4Represents a straight-chain or branched alkyl group having 1 to 4 carbon atoms, R2～R4May be the same or different from each other. Means with A1The bond of (2). )
In this case, R is preferably R1Is triisopropylsiloxymethyl represented by the following formula (3B).
The present invention also provides an apparatus for producing double-stranded DNA, which uses the above-mentioned DNAThe apparatus for producing a double-stranded DNA, which produces a double-stranded DNA having an adhesive end by using any one of the primers, comprising: a forward primer complementary to a part of the sequence of the antisense strand of the template DNA as a template and having a structure represented by the above formula (1); a reverse primer complementary to a part of the sequence of the sense strand of the template DNA and having a structure represented by the formula (1); an amplification device for performing a Polymerase Chain Reaction (PCR) for a plurality of cycles using the template DNA as a template to generate a forward-side extended strand extended by the forward primer and a reverse-side extended strand extended by the reverse primer, and annealing the forward-side extended strand and the reverse-side extended strand to generate a double-stranded DNA with a 3' -end recess; a smoothing device for smoothing the 3' -end of the double-stranded DNA with a Klenow fragment; and a deprotecting cleavage apparatus for cleaving R1Deprotection cleaves DNA at the moiety of formula (1) above to form an adhesive terminus protruding from the 3' terminus.
The present invention is also a method for producing a double-stranded DNA having an adhesive end using any one of the primers, comprising: a preparation step of preparing a forward primer that is complementary to a partial sequence of an antisense strand of a template DNA as a template and has a structure represented by formula (1), and a reverse primer that is complementary to a partial sequence of a sense strand of the template DNA and has a structure represented by formula (1); an amplification step of performing a Polymerase Chain Reaction (PCR) for a plurality of cycles using the template DNA as a template to generate a forward-side extended strand extended by the forward primer and a reverse-side extended strand extended by the reverse primer, and annealing the forward-side extended strand and the reverse-side extended strand to generate a double-stranded DNA having a 3' -end recess; a smoothing step of forming a smoothed end of the 3' -end of the double-stranded DNA by using a Klenow fragment; and a deprotection cleavage step of cleaving the above R1Deprotection cleaves DNA at the moiety of formula (1) above to form an adhesive terminus protruding from the 3' terminus.
In this case, R is as defined above1Is light divided into light represented by the above formula (2A)The protecting group to be deprotected and the cleavage step to be deprotected are preferably carried out by irradiating R mentioned above with light1And (4) deprotection.
Or, the above R1In order to deprotect the cleavage step for the fluoride-decomposable protecting group represented by the formula (2B), it is preferable that R is cleaved by fluoride1And (4) deprotection.
Effects of the invention
According to the present invention, a primer that can specifically cleave a single strand at a specific site and can be produced at low cost can be provided. Further, the present invention can provide an apparatus for producing a double-stranded DNA having an adhesive end and a method for producing a double-stranded DNA using such a primer.
Drawings
FIG. 1 is a schematic diagram showing a method and an apparatus for producing a double-stranded DNA having an adhesive end.
FIG. 2 is a diagram showing an experiment of a cleavage reaction of an oligonucleotide comprising a photocleavable analog.
FIG. 3 is a diagram showing an experiment using a cloning reaction in which an adhesive end is formed by a photocleavage reaction.
FIG. 4 is a graph showing the experimental results of an oligonucleotide cleavage reaction comprising a photocleaved analog.
FIG. 5 is a graph showing the experimental results of the cleavage reaction of an oligonucleotide comprising a fluorocleavable analog.
Detailed Description
1. Primer and method for producing the same
The primer of the present invention will be described below. The primer of the present invention is a primer for amplifying a nucleic acid, and has a structure represented by the following formula (1).
(Here, A)1represents-S-, -S-or-Se-, B represents a base group, R1Represents a decomposable protecting group. It means a bond to a sugar of an adjacent nucleotide. ). Furthermore, in the bond, the bond at the 3 'side of the formula (1) is that between the 3' side and the adjacent nucleotideThe 5 'carbon of the sugar is bonded, and the 5' side bond is bonded to the 3 'carbon of the sugar of the adjacent nucleotide at the 5' side.
B is a base, specifically, in the case of a DNA primer, B is selected from adenine, guanine, cytosine and thymine, and in the case of an RNA primer, B is selected from adenine, guanine, cytosine and uracil.
R1The degradable protecting group of (2) means a protecting group (substituent) which is degraded by any treatment. The processing described here may be exemplified by: light irradiation treatment, reduction treatment, alkali treatment, acid treatment, oxidation treatment, desilylation treatment, heat treatment, esterase treatment, phosphatase treatment, and the like.
(1) Photolytic protecting group
In the case where the treatment is light irradiation, R is preferably1Is a photolytic protecting group represented by the following formula (2A).
(Here, A)2Represents an alkylene group having 1 to 3 carbon atoms, A3Represents an alkylene group having 1 to 3 carbon atoms. Means with A1The bond of (2). )
Examples of the alkylene group having 1 to 3 carbon atoms include: methylene, ethylene, propylene.
In particular, R1A2-nitrobenzyloxymethyl group represented by the following formula (3A) is suitable.
(2) Fluoride-decomposable protecting group (silyl protecting group)
When the treatment is a reduction treatment, R is preferably1Is a fluoride-decomposable protecting group (silyl-type protecting group) represented by the following formula (2B).
(Here, A)4Represents an alkylene group having 1 to 3 carbon atoms, R2～R4Represents a straight-chain or branched alkyl group having 1 to 4 carbon atoms, R2～R4May be the same or different from each other. Means with A1The bond of (2). )
Examples of the straight-chain or branched alkyl group having 1 to 4 carbon atoms include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl.
In particular, R1Triisopropylsiloxymethyl group represented by the following formula (3B) is preferable.
(3) Other decomposable protecting groups
Examples of the decomposable protecting group which can be detached from the modified base by the base treatment include: isobutyryl, benzoyl, acetoxymethyl, and the like. Examples of the decomposable protecting group which can be detached from the modified base by acid treatment include: a trityl group. Examples of the decomposable protecting group which can be removed from the modified base by oxidation treatment include: allyloxymethyl, dimethoxybenzyloxymethyl, trimethoxybenzyloxymethyl and the like. Examples of the decomposable protecting group which can be detached from the modified base by desilylation include: t-butyldimethoxysiloxymethyl group, t-butyldiphenylsiloxymethyl group and the like. Examples of the decomposable protecting group which can be detached from the modified base by heat treatment include: an isocyanate group. Examples of the degradable protecting group which can be detached from the modified base by esterase treatment include: acetoxymethyl group. Examples of the degradable protecting group which can be detached from the modified base by phosphatase treatment include: methyl phosphate group.
The primer of the present invention is a single-stranded DNA or single-stranded RNA particularly suitable for use in PCR, and is an oligonucleotide or polynucleotide having a structure represented by the above formula (1) in a molecule. The number of base pairs of the primer can be appropriately set according to the sequence of the target DNA or the like, and is usually, for example, 20 base pairs or less in the oligonucleotide, and more than 20 base pairs in the polynucleotide. The upper limit of the number of base pairs in a polynucleotide is not particularly limited, and a primer generally used is preferably 40 base pairs or less, for example. The lower limit of the number of base pairs of the oligonucleotide is not particularly limited, and any length that can be used as a primer is sufficient, and a primer that is generally used is preferably 5 base pairs or more, for example.
2. Method for producing primer
The primer of the present invention can be produced by synthesizing a modified nucleoside (hereinafter, sometimes referred to as "nucleoside derivative") having a structure represented by the following formula (4), and then linking an unmodified nucleotide thereto by a solid phase synthesis method such as a phosphoramidite method.
(Here, A)1、R1As defined in the above formula (1), B means a base or a modified base. )
As an outline of the method for synthesizing the primer, first, the 3 '-hydroxyl group and the 5' -hydroxyl group of the nucleoside are protected with a linker element A1By substitution of the 2' hydroxy group with a decomposable protecting group R1And the A1Binding followed by deprotection of the 3 'and 5' hydroxyls. Thereafter, phosphoramidite or the like is reacted, and unmodified nucleotides are linked by a solid phase synthesis method to synthesize a primer. Specific methods for producing the primers listed in the examples will be described in detail below.
(a) Nucleoside derivatives 1 ([ color ] A) having photolytic protecting groups1is-S-radical, R1Compound which is 2-nitrobenzyloxymethyl) and synthesis of primers
The description is made according to the synthetic scheme described above. In the synthetic schemes described below, the numbers indicate the numbers of the compounds. First, a nucleoside (adenosine in the following scheme) was prepared as a starting material (compound 1). Next, 1, 3-dichloro-1, 1,3, 3-tetraisopropyl disiloxane is reacted with a nucleoside in a solvent such as pyridine. Thus, a cyclic structure of siloxane bond is formed between the 3 '-hydroxyl group and the 5' -hydroxyl group of ribose, and the hydroxyl groups at the 3 '-position and the 5' -position are protected (compound 2).
Next, N-phenyltrifluoromethanesulfonimide is added, and a nucleophilic reagent such as N, N-dimethyl-4-aminopyridine (DMAP) is reacted in a solvent such as Dichloromethane (DCM) to convert the 2' -position of ribose to a trifluoromethanesulfonic group (compound 3). Next, a compound having a thiol group such as potassium thioacetate is reacted in the presence of N, N-Dimethylformamide (DMF) to form a thioester at the 2' -position of ribose (compound 4). Then reacted in an ammonia/methanol solution to convert the thioester group to a thiol group (compound 5).
Then, 2-nitrobenzylchloromethyl ester, N-Diisopropylethylamine (DIPEA) and Tetrahydrofuran (THF) were added to bond the thiol to the 2-nitrobenzyloxymethyl group as a decomposable protecting group (Compound 6). Benzoyl chloride (BzCl) and pyridine were further added to bind the benzoyl group to the amino group of the base (compound 7). In this state, tetra-n-butylammonium fluoride (TBAF) and Tetrahydrofuran (THF) were added to remove protecting groups at the 3 '-and 5' -positions of ribose to form a hydroxyl group (Compound 8). By doing so, a nucleoside derivative (compound 8) can be synthesized.
Next, the nucleoside derivative is modified to prepare a primer by linking another nucleotide to the nucleoside derivative by the phosphoramidite method. First, 4 ' -dimethoxytrityl chloride (DMTrCl) and pyridine were added to the nucleoside derivative to bond the 4,4 ' -dimethoxytrityl chloride to the 5 ' -hydroxyl group of ribose (Compound 9). Next, 2-cyanoethyl-N, N-diisopropylchlorophosphidene was added to THF and DIPEA to bond the phosphoramidite to the 3' -hydroxyl group of ribose (Compound 10). Thereafter, the nucleotide is subjected to solid phase synthesis to synthesize a primer in such a manner as to become a desired sequence, using a conventional method.
(b) Nucleoside derivative 3 ([ color ] A) having a fluorolytic protecting group1is-S-radical, R1Compound being triisopropylsiloxymethyl) and synthesis of primers
The synthesis scheme up to compound 4 is the same as that of the synthesis of nucleoside derivative 1 described above. Next, 3 HF-Et was added3N (anhydrous Hydrogen Fluoride (HF), triethylamine (Et)3N) is 3: 1) and THF, etc. to remove the protecting groups at the 3 '-and 5' -positions of ribose to give hydroxyl groups (Compound 11). Then, DMTrCl and pyridine were added to bind 4,4 '-dimethoxytritylchloride to the 5' -hydroxyl group of ribose (Compound 12). Next, in ammonia methanol (NH)3MeOH) or DMF, and a silane compound such as chloromethyloxytriisopropylsilane (TOMCl) is reacted to bond triisopropylsiloxymethyl to the 2' -position of ribose (Compound 13).
Next, the nucleoside derivative is modified to prepare a primer by linking another nucleotide to the nucleoside derivative by the phosphoramidite method. First, chlorotrimethylsilane (TMSCl), BzCl, and pyridine were added to bind the benzoyl group to the amino group of the base and to bind the trimethylsilane to the 3' hydroxyl group of ribose (compound 14). Then, HF and pyridine are reacted with each other to form a hydroxyl group at the 3' -position of ribose (compound 15). Subsequently, as in the case of the synthesis of the nucleoside derivative 1, phosphoramidite is bonded to the 3' hydroxyl group of ribose (compound 16).
(c) Nucleoside derivatives 3 ([ color ] A) having photolytic protecting groups1is-Se-group, R1Compound which is 2-nitrobenzyloxymethyl) and synthesis of primers
Synthetic schemes up to Compound 3 and the nuclei described aboveThe same applies to the synthesis of glycoside derivative 1. Next, di-p-toluoyl selenide, piperidine, DIPEA are reacted in a solvent such as DMF (Compound 17). Then piperidine, Air (Air) and NaBH4And 2-nitrobenzylchloromethyl ester in a solvent such as DMF to bond the 2' -position of ribose to the selenium-based compound having a photolytic protecting group (Compound 18).
Thereafter, compounds 19 to 20 were synthesized in this order by treating them in the same manner as in the synthesis of nucleoside derivative 1. Then, a nucleotide is linked to the nucleoside derivative by the phosphoramidite method to produce a primer. This is also a process similar to the synthesis of the nucleoside derivative 1, and compound 21 and compound 22 are synthesized in this order.
3. Method and apparatus for producing double-stranded DNA having cohesive ends
Next, a method and an apparatus for producing a double-stranded DNA having an adhesive end will be described. The double-stranded DNA production apparatus of the present invention is an apparatus for producing a double-stranded DNA having an adhesive end using the primer of the present invention. The method for producing a double-stranded DNA of the present invention is a method for producing a double-stranded DNA having an adhesive end using a primer of the present invention, using a template DNA comprising a target DNA sequence. Hereinafter, description will be given with reference to fig. 1.
First, a primer set for PCR amplification including a forward primer and a reverse primer is prepared as a reagent (preparation step). The forward primer is complementary to a part of the sequence of the antisense strand of the template DNA and has the structure represented by formula (1). The reverse primer is complementary to a part of the sequence of the sense strand of the template DNA and has the structure represented by formula (1).
As shown in FIG. 1 (a), the forward primer and the reverse primer are sequenced in such a manner as to sandwich the target DNA sequence to be amplified. In addition, the position of the nucleotide having a decomposable protecting group of formula (1) in the primer is designed to be adjacent to the 5 ' side of the nucleotide on the most 5 ' terminal side on the 5 ' recess side in the sequence of the targeted adhesion terminus ((d) of the figure). That is, the nucleotide on the 3 'side adjacent to the nucleotide having the structure of formula (1) becomes the nucleotide at the 5' end of the cohesive end. Other reagents include polymerase used for PCR (Taq polymerase, etc.), buffer, dNTP, etc.
Next, the sequence of the template DNA is amplified using a PCR device (amplification means) (amplification step). The PCR apparatus performs a Polymerase Chain Reaction (PCR) for a plurality of cycles using the template DNA as a template to generate a forward-side extended strand extended by the forward primer and a reverse-side extended strand extended by the reverse primer, and anneals the forward-side extended strand and the reverse-side extended strand to generate a double-stranded DNA with a 3' -end notch ((b) of the figure).
In PCR, a sequence of a template DNA is amplified by repeating heat denaturation, annealing, and extension reactions. Although it depends on the PCR conditions, the thermal denaturation is performed at about 95 ℃ for 1 to 3 minutes, the annealing is performed at Tm. + -. 5 ℃ of the primer, and the extension reaction is performed for 1 to 10 minutes. The number of PCR cycles is not particularly limited, but is usually about 24 to 40 cycles.
As shown, a 3' -end-recessed double-stranded DNA was contained in the PCR amplification product. This is due to: when a complementary strand is synthesized using a primer as a template, the cleavable protecting group of formula (1) inhibits the polymerase reaction and stops the reaction.
Next, as shown in FIG. c, the 3' -end of the recess is made to be a smooth end by a filling-in reaction using Klenow Fragment (smoothing apparatus) (smoothing step). As reagents for this reaction, in addition to Klenow Fragment (enzyme), there can be mentioned: dNPT, buffer, etc. The conditions for the filling-in reaction may be set appropriately, for example, at 37 ℃ for 10 to 30 minutes, and then at 70 ℃ for 10 minutes to inactivate the enzyme.
Thereafter, as shown in (d), R is subjected to a predetermined treatment1Deprotection cleaves the single-stranded DNA at the structural part of formula (1) to form an adhesive end protruding from the 3' end (deprotected cleavage step). The prescribed treatment is for converting R1Examples of the deprotection treatment include: the light irradiation treatment and the reduction treatment.
Hereinafter, a cutting mechanism will be described. When a predetermined treatment is performed as shown in the following formula, the decomposable protecting group R of formula (1)1Detachment of hydrogen from the linker A1And (4) combining. Joint A1Form a heterocyclic ring consisting of three-membered rings with the 2 'carbon and 3' carbon of ribose (e.g.At A1Sulfur forms a thiirane ring), whereby the phosphate bond of the 3' carbon is cleaved.
Examples of the light irradiation treatment include: a method of irradiating light having a wavelength of 300 to 400nm for 1 to 30 minutes using a light source device (deprotecting dicing apparatus). Examples of the reduction treatment include: a method of treating the substrate with fluoride ions such as tetra-n-butylammonium fluoride (TBAF) at 70 to 80 ℃ for 1 to 30 minutes, for example. Thus, the primer is cleaved from the single-stranded DNA comprising the nucleoside of formula (1) on the 5 ' -end side, and a double-stranded DNA having a 3 ' -protruding end (5 ' -recessed end) can be synthesized. For other treatments, deprotection and cleavage of single-stranded DNA are performed in the same manner using a device for deprotecting a decomposable protecting group (deprotecting cleavage device).
In the present invention, the cohesive ends can be freely designed regardless of restriction enzymes or the like, and thus DNA having a desired sequence can be freely ligated. For example, the sequences of both the target DNA and the vector may be designed, and the both may be ligated by cleaving the both with deprotection to form a common cohesive end, thereby preparing a recombinant DNA, which may be used for cloning or library preparation, construction of a large-scale expression system, and the like. In addition, by ligating a plurality of genome sequences having cohesive ends, a genome overlap reaction can be performed in vitro. Alternatively, the genome stacking reaction may be carried out in a cell by introducing double-stranded DNA into the cell in a state of a smooth end and subjecting the double-stranded DNA to deprotection cleavage treatment in the cell.
Examples
The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, "%" is expressed on a mass basis (mass percentage) unless otherwise specified.
1. Photocleavage like (S)
1-1. Synthesis of photocleavable analog (ajo)
In the following, a synthetic scheme of photocleaved analogs is shown. The sequence of synthesis of the photocleavable analog is described below in terms of this synthesis scheme.
(1)3 ', 5' -O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) adenosine (Compound 2)
3.9mL (12.3mmol) of 1, 3-dichloro-1, 1,3, 3-tetraisopropyl disiloxane was added to a solution of 3.00g (11.2mmol) of adenosine (Compound 1) in 112mL of pyridine, and the reaction mixture was stirred at 0 ℃ for 1 hour. Thereafter, the reaction mixture was warmed to room temperature and stirred for 5.5 hours. The solvent was evaporated in vacuo using H2O and CH2Cl2The residue was extracted. The organic phase was washed with 1M HCl solution, saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The residue was chromatographed on silica gel (CH)2Cl2MeOH, 100/0-95/5 v/v) to yield 5.43g (10.65mmol) of Compound 2 (95%).
1H－NMR(400MHz、DMSO－d6)：δ0.95－1.07(m,8H),3.92(dd,J＝12.8,2.8Hz,1H),3.97－4.01(m,1H),4.05(dd,J＝12.8,3.2Hz,1H),4.51(d,J＝5.6Hz,1H),4.79(dd,J＝8.8,5.2Hz,1H),5.60(brs,1H),5.86(d,J＝1.2Hz,1H),7.30(brs,2H),8.06(s,1H),8.20(s,1H)；LRMS(ESI+)calc.m/z 510.26(M+H+),532.24(M+Na+),found m/z 510(M+H+),532(M+Na+)。
(2)3 ', 5 ' -O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2 ' -O-trifluoromethanesulfonyl-adenosine (Compound 3)
4.57g (12.8mmol) of N-phenyltrifluoromethanesulfonimide are added at 0 ℃ to a solution of 5.43g (10.7mmol) of Compound 2 and 3.90g (32.0mmol) of DMAP in 106mL of CH2Cl2In (1). The reaction mixture was stirred at 0 ℃ for 1 hour and at room temperature for 2 hours. With cooled 0.1M AcOH solution and CH2Cl2The mixture is extracted. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The resulting residue was purified by silica gel chromatography (hexane/EtOAc, 1/1v/v) to give 4.76g (7.42mmol) of Compound 3 (69%).
1H－NMR(400MHz,DMSO－d6)：δ0.98－1.11(m,28H),3.94－4.08(m,3H),5.37(dd,J＝9.6,5.6Hz,1H),6.08(d,J＝4.8Hz,1H),6.45(s,1H),7.42(br s,2H),8.03(s,1H),8.26(s,1H)；LRMS(ESI+)calc.m/z642.21(M+H+),664.19(M+Na+),found m/z 642(M+H+),664(M+Na+)。
(3) 9- [3, 5-O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2-deoxy-2-acetylthio-. beta. -D-arabinofuranosyl ] adenine (Compound 4)
1.06g (1.65mmol) of Compound 3 and 302mg (2.64mmol) of CH3COOSK is dissolved in 4.1mL DMF and stirred at room temperature for 20 hours. With saturated NaHCO3The reaction mixture was extracted with hexane/EtOAc (1/3 v/v). The organic phase was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The resulting residue was purified by silica gel chromatography (hexane/EtOAc, 2/1-1/2 v/v) to give 0.57g (1.00mmol) of Compound 4 (61%).
1H－NMR(400MHz,DMSO－d6)：δ1.01－1.18(m,28H),2.19(s,3H),3.89－3.98(m,2H),4.17(dd,J＝11.6,6.4Hz,1H),4.62(dd,J＝10,8.0Hz,1H),5.26(t,J＝8.0Hz,1H),6.39(d,J＝8.0Hz,1H),7.34(br s,2H),8.02(s,1H),8.10(s,1H)；HRMS(ESI+)calc.m/z 568.24(M+H+),590.23(M+Na+),found m/z 568.2467(M+H+),590.2286(M+Na+)。
(4) 9- [3, 5-O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2-deoxy-2-thio- β -D-arabinofuranosyl ] adenine (Compound 5)
To a solution of 2.11g (3.72mmol) of compound 4 in 25mL MeOH was added a catalytic amount of sodium methoxide in 28% MeOH. The mixture was stirred at room temperature for 4 hours with H2O and EtOAc extractionAnd (6) taking. H for organic phase2Washed with brine and dried (Na)2SO4) Concentration in vacuo afforded 1.96g (3.72mmol) of compound 5 (eq).
1H－NMR(400MHz,CDCl3)：δ1.04－1.14(m,21H),1.18(s,7H),3.81－3.92(m,2H),4.06(dd,J＝13.2,3.2Hz,1H),4.22(dd,J＝13.2,3.6Hz,1H),4.60－4.65(m,1H),5.64(s,1H),6.38(d,J＝7.2Hz,1H),8.10(s,1H),8.34(s,1H)。
(5) 9- [3, 5-O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2-deoxy-2- (2-nitrobenzyloxymethyl) thio-. beta. -D-arabinofuranosyl ] adenine (Compound 6)
1mL (1.30mmol) of a 1.3M solution of 2-nitrobenzyloxymethyl chloride was added to 0.53g (1.00mmol) of compound 5 and 524. mu.L (1.30mmol) of DIPEA in 4.0mL of CH at 0 deg.C2Cl2The solution was stirred for 1 hour. By H2O and CH2Cl2The mixture is extracted. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The residue was chromatographed on silica gel (CH)2Cl2MeOH, 50/1-20/1 v/v) to yield 0.57g (0.82mmol) of Compound 6 (82%).
1H－NMR(400MHz、CDCl3))：δ1.00－1.11(m 21H),1.17(s,7H),3.88－3.98(m,2H),4.04(dd,J＝12.8,2.8Hz,1H),4.19(dd,J＝13.2,4.4Hz,1H),4.67－4.84(m,5H),7.44－7.48(m,1H),7.62－7.72(m,2H),7.94(s,1H),8.07(dd,J＝8.0,1.2Hz,1H),8.23(s,1H)。
(6)N6-benzoyl-9- [3, 5-O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2-deoxy-2- (2-nitrobenzyloxymethyl) thio-beta-D-arabinofuranosyl]Adenine (Compound 7)
To a solution of 0.56g (0.81mmol) of Compound 6 in 5mL of pyridine was added 282. mu.L (2.43mmol) of benzoyl chloride, and the mixture was stirred at room temperature for 3 hours. The mixture was cooled to 0 ℃ and 2mL of H were added2O, the mixture was stirred for 0.5 hour. 2mL of concentrated NH was added3(aqueous solution), the mixture was stirred for 0.5 hour. By H2O and EtOAc extraction of the reaction mixture. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The resulting residue was purified by silica gel chromatography (hexane/EtOAc, 1/1v/v) to give 0.46g (0.58mmol) of Compound 7 (72%).
1H－NMR(400MHz,CDCl3)：δ1.01－1.10(m,21H),1.18(s,8H),3.93－4.09(m,2H),4.21(dd,J＝12.8,3.6Hz,1H),4.69－4.83(m,5H),7.46－7.56(m,3H),7.60－7.69(m,3H),7.96(dd,J＝8.0,1.2Hz,1H),8.05－8.11(m,2H),8.73(s,1H),8.79(dd,J＝5.2,1.6Hz,1H)。
(7)N6-benzoyl-9- [ 2-deoxy-2- (2-nitrobenzyloxymethyl) thio-beta-D-arabinofuranosyl]Adenine (Compound 8)
To a solution of 0.47g (0.59mmol) of Compound 7 in 5mL of THF was added 1.2mL of a 1M THF solution of TBAF, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated in vacuo and the resulting residue was chromatographed on silica gel (CH)2Cl2EtOAc, 50/1-20/1 v/v) to yield 0.33g (0.59mmol) of compound 8 (quantitative).
1H－NMR(400MHz,CDCl3)：δ3.16(d,J＝5.6Hz,1H),3.64－3.70(m,1H),3.73－3.78(m,1H),3.80－3.84(m,1H),3.97(dd,J＝7.2,2.8Hz,1H),4.38－4.45(m,1H),4.61－4.84(m,4H),5.12(t,J＝5.6Hz,1H),5.85(d,J＝6.0Hz,1H),6.62(d,J＝7.6Hz,1H),7.53－7.59(m,3H),7.62－7.67(m,2H),7.73－7.77(m,1H),8.02－8.06(m,3H),8.61(d,J＝4.8Hz,2H),11.11(s,1H)。
(8)N6-benzoyl-9- [ 5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2- (2-nitrobenzyloxymethyl) thio-beta-D-arabinofuranosyl]Adenine (Compound 9)
To a solution of 0.33g (0.59mmol) of Compound 8 in 4.0mL of pyridine was added 0.24g (0.71mmol) of 4, 4-dimethoxytrityl chloride. The reaction mixture was stirred at room temperature for 7.5 hours. By H2O and EtOAc extraction mixture. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) In aConcentrating in vacuum. The residue was chromatographed on silica gel (CH)2Cl2MeOH, 60/1-20/1 v/v) to yield 0.38g (0.44mmol) of Compound 9 (72%).
1H－NMR(400MHz,CDCl3)：δ1.04(d,J＝6.0Hz,5H),2.17(s,1H),3.18(d,J＝3.2Hz,1H),3.56(d,J＝4.4Hz,2H),3.78(d,J＝1.2Hz,6H),3.85(dd,J＝7.6,1.6Hz,1H),4.07－4.11(m,1H),4.62－4.71(m,3H),4.84(d,J＝12.Hz,1H),4.94(d,J＝14.4Hz,1H),6.65(d,J＝7.6Hz,1H),6.80－6.83(m,4H),7.20－7.33(m,4H),7.42－7.64(m,7H),8.03－8.06(m,3H),8.20(s,1H),8.72(s,1H),9.07(s,1H)。
(9)N6-benzoyl-9- { 3-O- [ 2-cyanoethoxy (diisopropylamino) phosphinyl alkyl]-5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2- (2-nitrobenzyloxymethyl) thio-. beta. -D-arabinofuranosyl } adenine (Compound 10)
241. mu.L (1.08mmol) of 2-cyanoethyl N, N-diisopropylchlorophosphamide was added to a solution of 0.42g (0.49mmol) of compound 9 and 417. mu.L (2.45mmol) of DIPEA in 2.5mL of THF, and the mixture was stirred at room temperature for 1 hour. With saturated NaHCO3The reaction mixture was extracted with EtOAc. The organic phase was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The resulting residue was purified by silica gel chromatography (hexane/EtOAc, 1/2v/v) to give 0.46g (0.49mmol) of Compound 10 (90%).31P－NMR(159MHz,CDCl3)：δ150.4,150.7。
1-2. Cleavage reactions of oligonucleotides comprising photocleavable analogs (A)
FIG. 2 is a diagram showing an experiment showing that the cleavage reaction of an oligonucleotide containing a photocleavable analog depends on light irradiation and heating. In the figure, (a) shows the oligonucleotide sequence and structure of 20 bases used and the predicted reaction mechanism, and (F) shows the modification with a fluorescein group. In this figure, (b) shows the results of gel electrophoresis analysis of the cleavage reaction with the modified polynucleotide, and (c) is a graph showing the results of quantification of the cleavage reaction product calculated from the results of electrophoresis shown in (b).
Mixing the oligonucleotides(5' fluorescein-d (ACGACTCA. multidot. CTATAGGGCGAA), 1. mu.M) was mixed in a mixture containing 10mM Tris-HCl (pH8.5), 50mM MgCl210mM Dithiothreitol (DTT).
40. mu.L of this solution was added to wells of a 96-well multi-well plate, and the concentration was adjusted to 4mW/cm using a MAX-305 light source device (light splitting toward the day)2The light quantity of (2) is irradiated with 365nm light for 0 minute, 1 minute, 5 minutes or 10 minutes. Then, each 10. mu.L of the solution was poured into a tube and heated at 72 ℃ for 0 minute, 5 minutes or 10 minutes. After cooling on ice, 10. mu.L of a 2 Xformamide sample solution (80 (v/v)% formamide, 50mM EDTA (pH8.0)) was added and analyzed by 20% modified polyacrylamide gel electrophoresis (PAGE) containing 7.5M urea. The oligonucleotides in the gel are labeled at the 5' end. Detection was performed using a ChemiDoc XRS + imaging system (Bio-Rad) based on fluorescein-based fluorescence.
At 4mW/cm2The amount of (C) was determined for 1. mu.M oligonucleotide, 10mM Tris-HCl (pH8.5), 50mM MgCl 240 μ L of 10mM DTT was irradiated with 365nm light for 5 minutes and heated at 72 ℃ for 5 minutes. Using a pipette tip filled with ZipTip. mu. -C18 resin, desalting and concentration were carried out, and the resulting product was sampled for MALDI-TOF molecular weight analysis using Ultraflex III (Bruker Daltonics). As the substrate, 3-hydroxypicolinic acid was used. The results of detection by MALDI-TOF MS of the raw material oligonucleotide (i) shown in FIG. a, its deprotected products (ii, iii) and cleavage products (iv, v) are shown in the following Table.
[ Table 1]
As shown in (b) and (c) of the figure, the number of cleavage products increased under the condition of 5 minutes of light irradiation, and the number of cleavage products further increased under the condition of 10 minutes of light irradiation. Further, as can be seen from fig. (b): in particular, under conditions of light irradiation for 10 minutes and heating time for 5 minutes or more, the band of the raw material becomes light, and the band of the cleavage product becomes dark, and it is found that: this condition is ideal for the cleavage reaction.
1-3. Using a cloning reaction in which an adhesive end is formed by photocleavage
Cloning and transformation will be described below with reference to FIG. 3. The method for preparing a GFP expression vector using a photocleaved analog is shown. To summarize, PCR reactions were first performed using pET21d and pacfp 1 as templates to obtain vector fragments and inserts. Then, after generating an adhesive fragment by light irradiation and heating, E.coli was transformed with a mixture of the both to obtain a ligation product in the form of plasmid DNA. The following description is made in detail.
The carrier-side fragment was modulated as follows. Using an Applied Biosystems 2720 thermal cycler, the reaction mixture [ 0.5. mu.M primer strands (pET21 d. mu.Fw and pET21 d. mu.Rev, Table 2), 0.8 ng/. mu.L pET21d (Novagen), 1 XPCR buffer for KOD-Plus-Neo, 1.5mM MgSO 1 XPCR buffer, 30 minutes/cycle (95 ℃, 30 seconds → 50 ℃, 30 seconds → 72 ℃) was prepared40.2mM dNTPs, 0.02U/. mu.L KOD-Plus-Neo (Toyobo)]. mu.L of restriction enzyme DpnI (Toyobo Co.) at 16U/. mu.L was added to 200. mu.L of the reaction solution after the PCR reaction, and the mixture was heated at 37 ℃ for 1 hour. A200. mu.L aliquot of TE-saturated phenol (Nacalai Tesque) and chloroform was added thereto, mixed vigorously, and centrifuged (14,000 Xg, 3 minutes) to separate the aqueous layer. Similarly, after the reaction solution was extracted with 200. mu.L of chloroform, 20. mu.L of 3M NaOAc (pH5.2) and 220. mu.L of isopropyl alcohol were added. After cooling at-30 ℃ for 1 hour, the DNA was recovered by centrifugation (20,000 Xg, 20 minutes). The target PCR product was purified by Agarose gel electrophoresis (0.8% Agarose S (and wako pure chemical industries) containing GelRed (and wako pure chemical industries)). DNA was extracted from the excised gel pieces (yield 1.27. mu.g DNA) using Wizard SV gel and PCR purification system (Promega).
The insert side segment is modulated as follows. Using an Applied Biosystems 2720 thermocycler, reaction mixtures [ 0.5. mu.M primer strands (pAcGFP 1. Fw and pAcGFP 1. Rev, Table 2), 0.8 ng/. mu.L pAcGFP1(Takara Bio), 1 XPCR buffer for KOD-Plus-Neo, 1.5mM MgSO 2 MgSO 1 XPCR buffer ] were prepared under conditions of (95 ℃, 30 seconds → 55 ℃, 30 seconds → 72 ℃,1 minute)/cycle and 30 cycles4、0.2mM dNTPs、0.02U/μL KOD－Plus－Neo)]. mu.L of 16U/. mu.L restriction enzyme D was added to 100. mu.L reaction solution after PCR reactionpnI (Toyobo), heated at 37 ℃ for 1 hour. To this solution, a 100. mu.L mixture of TE-saturated phenol (Nacalai Tesque) and chloroform was added, and the mixture was vigorously mixed and then centrifuged (14,000 Xg, 3 minutes), and the aqueous layer was separated. Similarly, after the reaction solution was extracted with 200. mu.L of chloroform, 10. mu.L of 3M NaOAc (pH5.2) and 110. mu.L of isopropyl alcohol were added. After cooling at-30 ℃ for 1 hour, the DNA was recovered by centrifugation (20,000 Xg, 20 minutes). The target PCR product was purified by Agarose gel electrophoresis (1.5% Agarose S (and wako pure chemical industries) containing GelRed (and wako pure chemical industries)). DNA was extracted from the excised gel pieces (yield 5.33. mu.g DNA) using Wizard SV gel and PCR purification system (Promega). The post-cleavage PCR primer sequences are shown in the following table. A in the sequence represents a photocleavable analog (FIG. 2).
[ Table 2]
mu.L of carrier fragment solution (6 ng/. mu.L of DNA, 10mM Tris-HCl (pH7.5), 5mM MgCl) was added to each well of a 96-well multi-well plate210mM DTT) and insert solution (8.4 ng/. mu.L DNA, 10mM Tris-HCl (pH7.5), 5mM MgCl210mM DTT), using a hand-held UV lamp (Funakoshi) at about 4mW/cm2365nm light was irradiated for 5 minutes. Then, 2 parts of the solution were recovered in 1.5mL tube and heated at 72 ℃ for 10 minutes. mu.L of 3M NaOAc (pH5.2) and 110. mu.L of isopropyl alcohol were added thereto, and the mixture was cooled at-30 ℃ for 1 hour and then centrifuged (20,000 Xg, 20 minutes) to recover the DNA. The DNA was dissolved in 5. mu.L of water, and 4.7. mu.L of the solution was added to 50. mu.L of E.coli competent cell solution (NEB 5-. alpha.competent E.coli (high efficiency), New England Biolabs). This was spread on LB agar medium containing 50. mu.g/mL ampicillin sodium, and cultured overnight at 37 ℃. Ligation products of 9 clones were obtained from the sample subjected to light irradiation and heating. In contrast, ligation products of 2 clones were obtained from the control sample which had not been irradiated with light and heated.
2. Fluorine cleaving analogs
2-1. Synthesis of fluoro-cleaving analogs (ajo)
Below, a synthetic scheme of the fluorine cleavage analog is shown. The sequence of synthesis of the fluoro cleavage analogs is illustrated below in accordance with this synthesis scheme.
(1) 9- [ 2-deoxy-2-thio- β -D-arabinofuranosyl ] adenine (Compound 11)
To a solution of 572mg (1.00mmol) of Compound 4 in 10mL of THF was added 409. mu.l (2.51mmol) of 3 HF-Et3N, the reaction mixture was stirred at room temperature for 2 hours. The mixture was concentrated in vacuo. The residue was chromatographed on silica gel (CHCl)3MeOH 92/8v/v) gave 265mg (0.81mmol) of Compound 11 (81%).
1H－NMR(400MHz,DMSO－d6)：δ2.15(s,3H),3.64－3.87(m,3H),4.34(dd,J＝10.0,7.6Hz,1H),4.50(dd,J＝16.8,10Hz,1H),5.12(t,J＝5.2Hz,1H),5.80(d,J＝6.4Hz,1H),6.42(d,J＝7.6Hz,1H),7.30(br s,2H),8.10(s,1H),8.22(s,1H)；HRMS(ESI+)calc.m/z 326.09(M+H+),348.07(M+Na+)found m/z 326.3798(M+H+),348.0800(M+Na+)。
(2) 9- [ 5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2-thio- β -D-arabinofuranosyl ] adenine (Compound 12)
To 265mg (0.815mmol) of Compound 11 in 8.0mL of pyridine was added 413mg (1.22mmol) of 4, 4' -dimethoxytrityl chloride, and the reaction mixture was stirred at room temperature for 2.5 hours. By H2O and CHCl3The reaction mixture was extracted. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The residue was chromatographed on silica gel (CHCl)3MeOH, 100/0-95/5 v/v) to yield 371mg (0.59mmol) of Compound 12 (72%).1H－NMR(400MHz,DMSO－d6)：δ2.17(s,3H),3.16(d,J＝8.4Hz,1H),3.45(dd,J＝10.4,7.6Hz,1H),3.69(s,3H),3.71(s,3H),4.02－4.07(m,1H),4.37(dd,J＝10.0,8.0Hz,1H),4.67(dd,J＝16.8,8.4Hz,1H),5.77(d,J＝5.6Hz,1H),6.47(d,J＝8.0Hz,1H),6.72(d,J＝8.8Hz,2H),6.78(d,J＝9.2Hz,2H),7.14－7.21(m,7H),7.29－7.33(m,4H),7.96(s,1H),8.14(s,1H)；13C－NMR(100MHz,DMSO)：δ30.0,51.2,54.9,63.7,72.2,82.5,83.3,85.4,112.9,113.0,119.1,126.5,127.6,129.6,129.7,135.4,135.5,140.1,144.8,148.7,152.3,156.0,157.9,158.0,193.9；HRMS(ESI+)calc.m/z 628.22(M+H+),650.20(M+Na+),found m/z 628.2216(M+H+),650.2054(M+Na+)。
(3) 9- [ 5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2-triisopropylsilyloxymethylthio-. beta. -D-arabinofuranosyl ] adenine (Compound 13)
To 749mg (1.19mmol) of Compound 12 in 6.0mL MeOH was added 28% NaOMe in 6.0mL MeOH. The reaction mixture was stirred at room temperature for 15 minutes and concentrated in vacuo. The residue is substituted by CH2Cl2Diluting with saturated NaHCO3The solution was washed with brine. The organic phase was dried (Na)2SO4) And concentrated in vacuo. The resulting residue was dissolved in 12mL THF without further purification. To the solution were added 1.4mL (8.33mmol) of DIPEA and 304. mu.L (1.31mmol) of (triisopropylsiloxy) methyl chloride, and the mixture was stirred at 0 ℃ for 16.5 hours. By H2O and CH2Cl2The reaction mixture was extracted. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The resulting residue was purified by silica gel chromatography (hexane/EtOAc, 1/1-1/2 v/v) to give 499mg (0.65mmol) of compound 13 (54%).1H－NMR(400MHz、DMSO－d6)：δ0.95－1.00(m,21H),3.19(d,J＝8.4Hz,1H),3.45(dd,J＝10.8,7.6Hz,1H),3.70(s,3H),3.71(s,3H),3.88(t,J＝7.6Hz,1H),3.97－4.01(m,1H),4.49(dd,J＝14.4,8.8Hz,1H),4.67(d,J＝10.4Hz,1H),4.76(d,J＝10.8Hz,1H),5.75(d,J＝6.0Hz,1H),6.49(d,J＝7.2Hz,1H),6.77(d,J＝8.8Hz,2H),6.81(d,J＝9.2Hz,2H),7.18－7.26(m,9H),7.35－7.36(m,2H),8.02(s,1H),8.11(s,1H)；13C－NMR(100MHz,DMSO)：δ11.2,17.6,17.7,52.5,54.9,63.5,63.8,74.2,82.9,84.0,85.4,113.0,118.7,126.6,127.7,129.6,135.4,135.5,139.4,144.8,149.0,152.3,155.9,157.9,158.0；HRMS(ESI+)calc.m/z 794.34(M+Na+),found m/z 794.3113(M+Na+)。
(4)N6－N6-dibenzoyl-9- [ 5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2-triisopropylsilyloxymethylthio-3-O-trimethylsilyl-beta-D-arabinofuranosyl]Adenine (Compound 14)
To a solution of 488mg (0.63mmol) of Compound 13 in 7.0mL of pyridine was added 96. mu.L (0.76mmol) of TMSCl at room temperature. After stirring for 1 hour, 218. mu.L (1.90mmol) of BzCl was added and the mixture was stirred at room temperature for 2 hours. 11mL of H was added to the reaction mixture2And O, stirring for 10 minutes. Then, 14mL of 28% NH was added3The aqueous solution was stirred for 15 minutes. By H2O and CH2Cl2The reaction mixture was extracted. With saturated NaHCO3The organic phase was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The resulting residue was purified by silica gel chromatography (hexane/EtOAc, 3/1-2/1 v/v) to give 211mg (0.20mmol) of Compound 14 (32%).
1H－NMR(400MHz、DMSO－d6)：δ－0.05(s,9H),0.95－1.05(m,21H),3.20(d,J＝8.8Hz,1H),3.39(dd,J＝11.2,6.8Hz,1H),3.68(s,3H),3.69(s,3H),3.97－4.02(m,2H),4.54－4.63(m,3H),6.68(d,J＝7.6Hz,1H),6.78(d,J＝3.6Hz,2H),6.81(d,J＝3.6Hz,2H),7.15－7.21(m,7H),7.32－7.34(m,2H),7.40(t,J＝8.0Hz,4H),7.55(t,J＝8.0Hz,2H),7.75(d,J＝7.2Hz,4H),8.60(s,1H),8.65(s,1H)；13C－NMR(100MHz,DMSO－d6)：δ0.3,11.2,17.6,17.7,52.2,55.0,65.8,69.5,74.3,82.7,84.7,85.6,113.1,126.6,127.1,127.7,128.9,129.6,129.7,133.3,133.4,135.2,135.5,144.4,146.1,150.8,151.6,152.4,158.0,178.9；HRMS(ESI+)calc.m/z1052.45(M+H+),found m/z 1052.4480(M+H+)。
(5)N 6－N 6Dibenzoyl-9- [ 5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2-triisopropylsilylalkoxymethylthio-beta-D-arabinofuranosyl]Adenine (Compound 15)
To a solution of 160mg (0.15mmol) of compound 14 in 1.5mL of pyridine was added 11. mu.L (0.42mmol) of 70% HF, and the reaction mixture was stirred at room temperature for 2 hours. By H2O and CH2Cl2The reaction mixture was extracted. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. The resulting residue was purified by silica gel chromatography (hexane/EtOAc, 2/1v/v) to give 93mg (0.09mmol) of compound 15 (62%).
1H－NMR(400MHz、DMSO－d6)：δ0.94－1.05(m,21H),3.20(d,J＝8.0Hz,1H),3.46(dd,J＝10.8,7.6Hz,1H),3.68(s,3H),3.69(s,3H),3.92－4.05(m,2H),4.41(dd,J＝15.2,8.8Hz,1H),4.65(d,J＝10.4Hz,1H),4.76(d,J＝10.8Hz,1H),6.64(d,J＝7.6Hz,1H),6.78(d,J＝8.0Hz,2H),6.80(d,J＝8.4Hz,2H),7.13－7.21(m,7H),7.31－7.33(m,2H),7.42(t,J＝7.6Hz,4H),7.57(t,J＝7.6Hz,2H),7.75(d,J＝7.6Hz,4H),8.54(s,1H),8.58(s,1H)；13C－NMR(100MHz,DMSO－d6)：δ11.2,17.6,17.7,52.5,54.9,63.6,66.0,74.1,83.0,84.8,85.4,113.0,126.6,127.1,127.7,128.9,129.5,129.8,133.3,133.5,135.2,135.8,144.6,146.0,150.8,151.6,152.4,152.9,158.0,171.9；HRMS(ESI+)calc.m/z 980.41(M+H+),found m/z 980.4331(M+H+)。
(6)N6－N6-dibenzoyl-9- { 3-O- [ 2-cyanoethoxy (diisopropylamino) phosphinyl alkyl]-5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2-triisopropylsilyloxymethylthio-. beta. -D-arabinofuranosyl } adenine (Compound 16)
To a solution of 93mg (0.01mmol) of Compound 15 in 950. mu.L of THF were added 42. mu.L (0.19mmol) of 2-cyanoethyl N, N-diisopropylchlorophosphamide and 80. mu.L (0.47mmol) of DIPEA, and the mixture was stirred at room temperature for 1 hour. By H2O and EtOAc extraction of the reaction mixture. The organic phase was saturated NaHCO3The solution was washed with brine and dried (Na)2SO4) And concentrated in vacuo. Passing the obtained residue through siliconPurification by gel chromatography (hexane/EtOAc, 2/1v/v) afforded 83mg (0.07mmol) of compound 16 (70%).
31P－NMR(159MHz、DMSO－d6)：δ149.1、149.9、HRMS(ESI+)calc.m/z 1180.52(M+H+)、found m/z 1180.5363(M+H+)。
2-2. Synthesis of oligonucleotides
Oligonucleotides were synthesized according to the phosphoramidite method using a DNA synthesizer. The 5' -end was fluorescently labeled with 6-FAM imide (Chemsenes). The imide of each of the synthesized cleavage analogs was prepared as an acetonitrile solution with a final concentration of 50mM, and introduced into the DNA primer using a DNA synthesizer. Deprotection was performed according to the conventional method, and each DNA was purified by 20% PAGE, and the structure was confirmed by using MALDI-TOF/MS (Bruker). The sequences of the synthesized DNAs are shown in the following Table.
[ Table 3]
3-3. Cleavage reactions of oligonucleotides comprising fluoro-cleaved analogs
Mixing the oligonucleotide (5' fluorescein-d ACGACTCA. multidot. CTATAGGGCGAA) and 1. mu.M) in a mixture containing 10mM Tris-HCl (pH8.5) and 50mM MgCl210mM Dithiothreitol (DTT), and then an equal amount of 1M TBAF in THF was added, and the mixture was heated at 72 ℃ for 10 minutes. After cooling on ice, the solvent was removed by a centrifugal evaporator, and the mixture was dissolved again in 10. mu.L of a 2 Xformamide sample addition solution (80 (v/v)% formamide, 50mM EDTA (pH 8.0)). The resulting samples were analyzed by 20% modified polyacrylamide gel electrophoresis (PAGE) with 7.5M urea. The oligonucleotides in the gel are labeled at the 5' end. Detection was performed using a ChemiDoc XRS + imaging system (Bio-Rad) based on fluorescein-based fluorescence. Fig. 4 shows a photograph of an electrophoresis gel. As shown in the figure, it was found that the oligonucleotide was cleaved by the cleavage reaction.
3. Photocleavable analog (Se)
3-1. Synthesis of photocleaved Se analog (ASe)
In the following, a synthetic scheme of photocleaved Se analogs is shown. The sequence of synthesis of the photocleavable analog is described below in terms of this synthesis scheme.
(1) 9- [3, 5-O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2-deoxy-2-p-toluoylseleno-beta-D-arabinofuranosyl ] adenine (Compound 17)
To a solution of 14.8g (46.7mmol) of di-p-toluoylseleno ether in 160mL of DMF were added 4.3mL (43.7mmol) of piperidine and 8.2mL (46.7mmol) of DIPEA, and the mixture was stirred at room temperature. After stirring for 5 minutes, 10g (15.6mmol) of Compound 3 was added to the above mixture, and the mixture was stirred at 60 ℃ for 1 hour. AcOEt was added to the reaction mixture, and 1N aqueous HCl, saturated NaHCO, was used3And (4) washing with an aqueous solution. Washing with brine and Na2SO4Drying to evaporate it to give crude compound. The crude product was purified by silica gel column chromatography (Hex/AcOEt ═ 1/1 to 1/4, v/v) to yield 5.7g of compound 17 (53%).
1H－NMR(400MHz,CDCl3)：δ8.16(1H,s),7.92(1H,s),7.56(2H,d,J＝8.4Hz),7.15(2H,d,J＝8.4Hz),6.47(1H,d,J＝7.6Hz),6.09(2H,brs),5.25(1H,dd,J＝10.0,8.0Hz),4.72(1H,dd,J＝10.4,7.6Hz),4.27(1H,dd,J＝12.0,4.8Hz),4.03－4.01(2H,m),2.34(3H,s),1.18－0.99(28H,m)；13C－NMR(100MHz,CDCl3)：δ192.5,155.2,151.9,149.4,145.3,140.2,135.5,129.6,127.5,120.1,85.0,84.3,73.5,62.1,50.4,21.8,17.6,17.5,17.4,17.3,17.1,13.7,13.2,12.9,12.5；77Se－NMR(75MHz,CDCl3)：δ535.6；HRMS(ESI+)calc.m/z 692.2197[M+H]+,found m/z 692.2200[M+H]+。
(2) 9- [3, 5-O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2-deoxy-2- (2-nitrobenzyloxymethyl) seleno-beta-D-arabinofuranosyl ] adenine (Compound 18)
Under the condition of air, underTo a solution of 400mg (0.58mmol) of Compound 17 in 4mL of DMF was added 143. mu.L (1.4mmol) of piperidine, and the reaction mixture was stirred at room temperature for 16 hours. The atmosphere was then exchanged with argon, the solution was aerated with argon for 10 minutes, and then 53mg (1.4mmol) NaBH was added4And stirred at room temperature. After 15 minutes, the reaction mixture was cooled to 0 ℃ and 0.87mmol of 2-nitrobenzyloxymethyl chloride in 2mL of DMF was added and stirred at 0 ℃ for 1 hour. By addition of H2Quenching the reaction with O, extracting with AcOEt, washing with brine, and washing with Na2SO4Drying to evaporate it. The crude product was purified by column chromatography on silica gel (CHCl)3Purification with MeOH 25/1, v/v) yielded 226mg of compound 18 (53%).
1H－NMR(400MHz,CDCl3)：δ8.14(1H,s),8.01－7.86(2H,m),7.68－7.32(3H,m),6.37(1H,d,J＝7.6Hz),6.15(2H,brs),5.02(1H,s,diastereotopic),4.97(1H,s,diastereotopic),4.86(1H,dd,J＝10.0,8.0Hz),4.77(1H,s,diastereotopic),4.75(1H,s,diastereotopic),4.13(1H,dd,J＝12.8,4.4Hz),4.01－3.94(2H,m),3.84－3.82(1H,m),1.11－0.95(28H,m)；13C－NMR(100MHz,CDCl3)：155.8,152.8,149.6,147.4,139.2,133.7,133.5,129.1,128.4,124.8,119.6,84.8,84.1,74.9,69.5,67.9,61.6,48.7,17.6,17.5,17.4,17.2,17.1,17.0,13.7,13.1,13.0,12.6；77Se－NMR(75MHz,CDCl3)：δ231.7；HRMS(ESI+)calc.m/z 739.2204[M+H]+,found m/z 739.2208[M+H]+。
(3)N6,N6-dibenzoyl-9- [3, 5-O- (1,1,3, 3-tetraisopropyl-1, 3-disiloxanediyl) -2-deoxy-2- (2-nitrobenzyloxymethyl) seleno-beta-D-arabinofuranosyl-l]Adenine (Compound 19)
To a solution of 200mg (0.27mmol) of compound 18 in 3mL of pyridine was added 125. mu.L (1.08mmol) of benzoyl chloride, and the mixture was stirred at room temperature for 4 hours. AcOEt was added to the reaction mixture with H2Washing with sodium chloride and Na2SO4Drying to evaporate it. The crude product was purified by silica gel column chromatography (hexane/AcOEt ═ 2/1-1/2, v/v) to give 126mg of compound 19 (49%).
1H－NMR(400MHz,CDCl3)：δ8.54(1H,s),8.13(1H,s),8.04－7.28(14H,m),6.47(1H,d,J＝7.6Hz),5.05－4.79(5H,m),4.19－3.85(4H,m),1.13－0.95(28H,m)；13C－NMR(100MHz,CDCl3)：δ172.3,152.9,152.0,151.9,147.6,143.8,134.2,133.7,133.2,133.0,129.6,129.3,128.7,128.6,128.0,124.9,85.4,84.3,75.2,69.7,68.2,61.7,49.1,17.6,17.5,17.4,17.2,17.1,14.2,13.7,13.1,13.0,12.6；77Se－NMR(75MHz,CDCl3)：δ234.4；HRMS(ESI+)calc.m/z947.2729[M+H]+,found m/z 947.2727[M+H]+。
(4)N6,N6-dibenzoyl-9- [ 2-deoxy-2- (2-nitrobenzyloxymethyl) seleno-beta-D-arabinofuranosyl]Adenine (Compound 20)
To a solution of 125mg (0.13mmol) of compound 19 in 1mL of THF was added 290. mu.L of a 1M THF solution of TBAF, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo and the resulting residue was purified by column chromatography on silica gel (CHCl3/MeOH 25/1, v/v) to give 65mg of compound 20 (71%).
1H－NMR(400MHz,CDCl3)：δ8.56(1H,s),8.51(1H,s),8.02(1H,d,J＝8.0Hz),7.81(4H,d,J＝7.2Hz),7.60－7.30(9H,m),6.55(1H,d,J＝7.6Hz),5.02(2H,s),4.83－4.69(3H,m),3.98－3.75(4H,m)；13C－NMR(100MHz,CDCl3)：δ172.4,152.6,152.1,151.9,147.7,144.6,133.9,133.8,133.2,132.7,130.1,129.5,128.9,128.4,127.5,125.0,86.2,85.1,73.5,69.5,68.5,59.9,49.7；77Se－NMR(75MHz,CDCl3)：δ250.1。
(5)N 6,N 6-dibenzoyl-9- [ 5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2- (2-nitrobenzyloxymethyl) seleno-beta-D-arabinofuranosyl]Adenine (Compound 21)
To a solution of 60mg (0.085mmol) of compound 20 in 0.8mL of pyridine was added 35mg (0.102mmol) of 4, 4' -dimethoxytrityl chloride. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with AcOEt and saturated H2And O washing. By usingNaHCO3Washing with aqueous solution, brine, and Na2SO4Drying to evaporate it. The crude product was purified by silica gel column chromatography (hexane/AcOEt ═ 1/1, v/v) to give 48mg of compound 21 (56%).
1H－NMR(400MHz,CDCl3)：δ8.52(1H,s),8.21(1H,s),8.03(1H,d,J＝8.0Hz),7.83－7.81(4H,m),7.57－7.15(18H,m),6.79－6.77(4H,m),6.60(1H,d,J＝7.2Hz),5.04－4.72(4H,m),4.66(1H,dd,J＝8.8Hz),4.05－4.03(1H,m),3.93(1H,dd,J＝8.8,7.2Hz),3.72(6H,s),3.52－3.50(2H,m)；13C－NMR(100MHz,CDCl3)：δ172.3,158.7,152.7,152.2,151.9,147.8,144.4,143.8,135.7,135.6,134.2,133.8,133.0,132.5,130.1,129.5,129.0,128.7,128.3,128.0,127.0,125.1,113.3,86.9,85.4,83.3,76.5,69.5,68.6,62.9,55.3,49.6；77Se－NMR(75MHz,CDCl3)：δ252.0；HRMS(ESI+)calc.m/z 1007.2513[M+H]+,found m/z 1007.2536[M+H]+。
(6)N 6,N 6-dibenzoyl-9- { 3-O- [ 2-cyanoethoxy (diisopropylamino) phosphinyl alkyl]-5-O- (4, 4' -dimethoxytrityl) -2-deoxy-2- (2-nitrobenzyloxymethyl) seleno-beta-D-arabinofuranosyl } adenine (Compound 22)
0.5mL of CH in 45mg (0.045mmol) of Compound 21 and 47. mu.L (0.27mmol) of DIPEA2Cl2To the solution was added 25. mu.L (0.11mmol) of 2-cyanoethyl N, N-diisopropylphosphoramidite, and the mixture was stirred at 0 ℃ for 1 hour. The reaction mixture was diluted with AcOEt and saturated NaHCO3Washing with aqueous solution and brine, and adding Na2SO4Drying to evaporate it. The crude product was purified by silica gel column chromatography (hexane/AcOEt ═ 1/1, v/v) to give 41mg of compound 21 (76%).
31P－NMR(159MHz,CDCl3)：δ150.6,150.3；HRMS(ESI+)calc.m/z 1207.3592[M+H]+,found m/z 12707.3624[M+H]+。
3-2. Synthesis of oligonucleotides
The synthesized cleavage analog ASeOf (a) imideThe amine was prepared as an acetonitrile solution with a final concentration of 50mM, and DNA oligomer was synthesized according to the phosphoramidite method using a DNA synthesizer. Deprotection was performed according to a conventional method by reverse phase HPLC [ Lachrom Elite manufactured by Hitachi High-Tech Science Co., Ltd.; chromatographic column, Hydrosphere C18 (250X 10mm) made by YMC]Purification and confirmation of structure using MALDI-TOF/MS (Bruker). The sequences of the synthesized DNAs are shown in the following Table.
[ Table 4]
| Sequence of |
| 5’-ACGACTCASeCTATAGGGCGAATTCGAGCTCGGT-3’ |
3-3. containing photocleavable analog (A)Se) Cleavage reaction of the oligonucleotide of (3)
In the presence of 10mM Tris-HCl (pH8.5), 5mM MgCl2After mixing 3. mu.M of oligonucleotide in 10mM Dithiothreitol (DTT) buffer, the mixture was irradiated with light MAX-305 (split into light in the daytime) at about 4mW/cm2Light having a wavelength of 365nm was irradiated for 10 minutes. The resulting sample was diluted 2-fold with a formamide sample solution (80 (v/v)% formamide, 50mM EDTA (pH8.0)), and analyzed by 15% modified polyacrylamide gel electrophoresis (PAGE) containing 7.5M urea. Oligonucleotides in the gel were stained with SYBR Green II nucleic acid gel stain and detected using a ChemiDoc XRS + imaging system (Bio-Rad). The results are shown in FIG. 5. From the results, it is found that: cleavage products were visualized by light irradiation in buffer at room temperature for 10 min.
Claims (9)
1. A primer, characterized by: which is a primer for amplifying a nucleic acid and has a structure represented by the following formula (1),
here, A1represents-S-, -S-or-Se-, B represents a base group, R1The term "degradable protecting group" means a bond to a sugar of an adjacent nucleotide.
2. The primer according to claim 1, characterized in that: the R is1Is a photolytic protecting group represented by the following formula (2A),
here, A2Represents an alkylene group having 1 to 3 carbon atoms, A3Represents an alkylene group having 1 to 3 carbon atoms, wherein A represents1The bond of (2).
3. The primer according to claim 2, characterized in that: the R is1Is a 2-nitrobenzyloxymethyl group represented by the following formula (3A),
4. the primer according to claim 1, characterized in that: the R is1Is a fluoride-decomposable protecting group represented by the following formula (2B),
here, A4Represents an alkylene group having 1 to 3 carbon atoms, R2～R4Represents a straight-chain or branched alkyl group having 1 to 4 carbon atoms, R2～R4May be the same or different from each other, means the same as A1The bond of (2).
5. The primer of claim 4, wherein: the R is1Is a triisopropylsiloxymethyl group represented by the following formula (3B),
6. a double-stranded DNA manufacturing apparatus for manufacturing a double-stranded DNA having an adhesive end using the primer according to any one of claims 1 to 5, comprising:
a forward primer complementary to a part of the sequence of the antisense strand of the template DNA as a template and having a structure represented by the formula (1);
a reverse primer complementary to a part of the sequence of the sense strand of the template DNA and having the structure represented by the formula (1);
an amplification apparatus that performs a plurality of cycles of Polymerase Chain Reaction (PCR) using the template DNA as a template to generate a forward-side extended strand extended by the forward primer and a reverse-side extended strand extended by the reverse primer, and anneals the forward-side extended strand and the reverse-side extended strand to generate a double-stranded DNA with a 3' end being recessed;
a smoothing device for forming a smoothed end of the 3' end of the double-stranded DNA using a Klenow fragment; and
deprotecting the cleavage apparatus of the R1Deprotection cleaves DNA at the moiety of formula (1) to form an adhesive terminus protruding from the 3' terminus.
7. A method for producing a double-stranded DNA, which is a method for producing a double-stranded DNA having an adhesive end using the primer according to any one of claims 1 to 5, comprising:
a preparation step of preparing a forward primer that is complementary to a partial sequence of an antisense strand of a template DNA as a template and has a structure represented by formula (1), and a reverse primer that is complementary to a partial sequence of a sense strand of the template DNA and has a structure represented by formula (1);
an amplification step of performing a Polymerase Chain Reaction (PCR) for a plurality of cycles using the template DNA as a template to generate a forward-side extended strand extended by the forward primer and a reverse-side extended strand extended by the reverse primer, and annealing the forward-side extended strand and the reverse-side extended strand to generate a double-stranded DNA with a 3' -end recess;
a smoothing step of forming a smoothed end of the 3' end of the double-stranded DNA by using a Klenow fragment; and
a deprotection cleavage step of converting the R1Deprotection cleaves DNA at the moiety of formula (1) to form an adhesive terminus protruding from the 3' terminus.
8. The method for producing a double-stranded DNA according to claim 7, wherein: the R is1A cleavage step of deprotecting the photodegradable protecting group represented by the formula (2A) by irradiating the group R with light1And (4) deprotection.
9. The method for producing a double-stranded DNA according to claim 7, wherein: the R is1A deprotection cleavage step of cleaving the R group with a fluoride for the fluoride-decomposable protecting group represented by the formula (2B)1And (4) deprotection.
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| WO2009113709A1 (en) * | 2008-03-11 | 2009-09-17 | 国立大学法人東京大学 | Method of preparing dna fragment having sticky end |
| CN103305498A (en) * | 2012-03-06 | 2013-09-18 | 中国科学院上海生命科学研究院 | Method for producing preset cohesive end at double-stranded DNA fragment end |
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| CN101434978A (en) * | 2007-11-14 | 2009-05-20 | 富士胶片株式会社 | Nucleic acid amplification method |
| WO2009113709A1 (en) * | 2008-03-11 | 2009-09-17 | 国立大学法人東京大学 | Method of preparing dna fragment having sticky end |
| CN103305498A (en) * | 2012-03-06 | 2013-09-18 | 中国科学院上海生命科学研究院 | Method for producing preset cohesive end at double-stranded DNA fragment end |
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| Title |
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| H OZAKI等: "Synthesis of oligodeoxyribonucleotides bearing a functional group at 2\'-position by post-synthetic modification", 《NUCLEIC ACIDS SYMP SER . 》, no. 42, pages 47 - 48 * |
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